Short-Chain Acyl-CoA Dehydrogenase Deficiency: Studies in a Large Family Adding to the Complexity of the Disorder
,
* Maxima Medisch Centrum, Veldhoven, The Netherlands
Academic Medical Center, University of Amsterdam, Department of Clinical Chemistry and Division of Emma’s Children’s Hospital, Amsterdam, The Netherlands
Research Institute for Molecular Medicine, Faculty of Health Sciences and Aarhus University Hospital, Aarhus N, Denmark
 |
ABSTRACT |
|---|
 TOP ABSTRACT METHODSDISCUSSIONREFERENCES |
|---|
Objective. To understand the expanding clinical and biochemical spectrum of short-chain acyl-CoA dehydrogenase (SCAD) deficiency, the impact of which is not fully understood.
Study Design. We studied a family with SCAD deficiency and determined urinary ethylmalonic acid excretion, plasma C4-carnitine, SCAD enzyme activity in fibroblasts and lymphocytes, DNA mutations in the SCAD gene, and clinical expression. The index patient was born prematurely and had otherwise unexplained cholestasis and hepatomegaly during the first year of life. His mother developed a hemolysis-elevated liver enzymes-low platelets (HELLP) syndrome while pregnant with the index patient.
Results. Two siblings had a homozygous inactivating 1138C>T mutation, whereas the father was compound heterozygous for this mutation and the common 625G>A polymorphism. There was a good correlation between the type of SCAD mutation, the residual SCAD enzyme activity, and the levels of urinary ethylmalonic acid and plasma C4-carnitine in each of the eight family members. Retrospective acylcarnitine analysis of the index patient’s Guthrie screening card confirmed the abnormal increase of C4-carnitine, suggestive of SCAD deficiency. None of the family members had hypotonia, developmental delay, or episodes of ketotic hypoglycemia.
Conclusion. Homozygosity for an inactivating SCAD mutation does not necessarily result in disease. The previously held opinion that SCAD deficiency is always a serious disorder may have been influenced by a clinical bias. Homozygosity for an inactivating 1138C>T SCAD mutation was assessed by neonatal screening of blood spot acylcarnitines. SCAD deficiency may be associated with maternal HELLP syndrome.
Key Words: mitochondrial fatty acid oxidation • short-chain acyl-CoA dehydrogenase • ethylmalonic acid • butyrylcarnitine • inactivating mutations
Abbreviations: SCAD, short-chain acyl-CoA dehydrogenase • EMA, ethylmalonic acid • HELLP, hemolysis-elevated liver enzymes-low platelets
Short-chain acyl-CoA dehydrogenase (SCAD) is an enzyme involved in mitochondrial short-chain fatty acid oxidation. It is active with fatty acids having chain lengths from C4 to C8 with a maximum activity toward C4. Twenty patients with SCAD deficiency have been reported,1 but at least 50 more have been identified (N. Gregersen, personal communication). The clinical presentation is characterized by hypotonia, developmental delay, seizures, microcephaly, lethargy, scoliosis, and finally a combination of hypoglycemia, vomiting, poor feeding, and failure to thrive.2,3 The biochemical presenting signal is a variably increased urinary ethylmalonic acid (EMA) excretion,4 occasionally with increased methylsuccinic acid and butyrylglycine; plasma acylcarnitine examination may show an increase of C4-carnitine (butyrylcarnitine). The SCAD activity in fibroblasts is either low or reduced (from undetectable to 40% of control). Mutation analysis in 14 reported cases showed 10 pathogenic mutations, among which was the 1138C>T missense mutation.1 In addition to pathogenic or inactivating mutations, two polymorphisms have been identified, 625G>A and 511C>T, which are common (14%) in the general population.5 Among the cases reported, a considerable number were shown to be heterozygous or homozygous for one of the polymorphisms. One patient with 625G>A homozygosity presented with maternal acute fatty liver of pregnancy.6 The precise relationship between the nature of the mutation/variation, the SCAD activity in cultured fibroblasts, and the clinical phenotype has not yet been established, and other factors may contribute to the actual consequences of certain mutations with respect to clinical signs and symptoms.7 It is still uncertain whether adverse cellular and physiologic conditions are sufficient to provoke clinical disease. Hence, understanding of the natural spectrum of SCAD deficiency is warranted.
In this report we add further data to the complexity of SCAD deficiency, including the observation that severe SCAD deficiency is not always disease-causing and that it may be associated with hemolysis-elevated liver enzymes-low platelets (HELLP) syndrome. Part of the data were presented previously in abstract form.8
|
METHODS |
|---|
|
TOPABSTRACTMETHODSDISCUSSIONREFERENCES |
|---|
Organic acids in urine were analyzed by gas chromatography/mass spectrometry of the methoxime/trimethylsilyl derivatives. Plasma acylcarnitines were analyzed as their butyl esters with electrospray tandem mass spectrometry.9 The activity of short-chain acyl-CoA dehydrogenase in lymphocytes or fibroblasts was measured using a high-performance liquid chromatography method. Cell lysates were incubated at pH 8 with butyryl-CoA and isovaleryl-CoA. Flavin adenine dinucleotide was added as a cofactor, and ferricenium was used as an artificial electron acceptor. As a consequence of the presence of native crotonase in the lysate, the formed enoyl-CoA esters were converted to the
-3-hydroxyacyl-CoA esters. The latter products were analyzed by reversed-phase high-performance liquid chromatography and UV detection after an incubation at 37°C for 30 minutes. The addition of excess isovaleryl-CoA was necessary to eliminate the conversion of butyryl-CoA to crotonyl-CoA by isovaleryl-CoA dehydrogenase. Details of the method will be published elsewhere. Mutation analysis of genomic DNA was performed by sequencing the SCAD gene as reported previously.1
Case Report
The index patient was the seventh child of healthy, first-degree consanguineous Turkish parents. He was their first son (Fig. 1). Their first child, a girl, died at home in Turkey at the age of 1 week after an uneventful pregnancy and delivery. Their other children were born in The Netherlands, all after uncomplicated pregnancies and deliveries. They are all in good mental and physical health.
|
The father is the youngest of eight children, three of his siblings, one brother and two sisters, died in Turkey before the age of 1 year; no further information was available, especially about the possible consanguinity of the father’s parents. However, they were from the same village.
The pregnancy with the index patient was complicated by hypertension and HELLP syndrome and premature delivery at 29 3/7 weeks. Birth weight was 930 g (5th percentile for gestational age); Apgar scores were 5 and 7 after 1 and 5 minutes, respectively. Because of respiratory distress he had to be ventilated for 10 days and had nasal continuous positive airway pressure for 4 weeks. Because of chronic lung disease he had low-flow oxygen for a total of 5 months. Neither hypoglycemia nor metabolic acidosis were documented in the neonatal period or afterward at routine controls or during metabolic stress associated with febrile illness or operation.
At the age of 2 months, hepatomegaly and jaundice with grossly elevated conjugated serum bilirubin was observed and diagnostic investigations were started. The stools were normally colored. Ultrasound examination confirmed an enlarged liver and spleen without structural abnormalities of the liver, spleen, and intra- or extrahepatic bile duct system, including the gallbladder. The results of hepatobiliary scintigraphy were normal. Serologic investigations for hepatitis A and B, cytomegalovirus, Epstein-Barr virus, toxoplasmosis, rubella, syphilis, and brucellosis were negative, as were cultures of feces and urine. Free thyroxine was 16 pmol/L (controls: 9.8–25.8), 
1-antitrypsin was 1.59 g/L (controls: 1.3–2.7), aspartate transaminase was 150 units/L (controls: 7–43), alanine transaminase was 40 units/L (controls: 3–47), lactate dehydrogenase was 390 units/L (controls: 26–534), and alkaline phosphatase was 1055 units/L (controls: 19–384). For bilirubin, indirect was 140 µmol/L (controls: 3–14), and direct was 102 µmol/L (controls < 5).
Organic acid analysis of a 24-hour urine showed a high EMA excretion, 124 to 380 mmol/mol creatinine (control children <18). Plasma acylcarnitine analysis showed C4-carnitine to be 4.7 µmol/L (controls <0.58 µmol/L). Fibroblast SCAD activity was 0.10 nmol/min/mg protein [control values (mean (range)): 0.48 (0.3–0.62)]. DNA analysis of the SCAD gene showed a homozygous 1138C>T mutation.
The neonatal screening card of the index patient was retrospectively examined for acylcarnitines and showed a marked increase of the C4-carnitine (3.5 µmol/L; controls < 0.87).
At the age of 3 years the boy was growing along the 3rd percentile (87 cm) for Turkish boys. The hepatosplenomegaly had resolved, and serum bilirubin and liver functions had normalized. His behavior was very active and he had a slight hypotonia. His mental development was considered to be normal.
Family Studies
Family examinations, including metabolic screening and DNA analysis, were conducted as shown in Table 1. The father, mother, and all their daughters were in good mental health, achieved normal school grades, and showed no abnormalities at physical examination.
|
Urinary EMA excretion was measured several times: in the father (I.1) EMA was in the upper normal range (12–14 mmol/mol creatinine; adult controls <12), in the eldest daughter (II.2) EMA was elevated (25–58 mmol/mol creatinine). All other family members had normal EMA levels in urine. Plasma C4-carnitine was increased in the father, I.1 (1.7–2.2 µmol/L; controls < 0.58), in the eldest daughter, II.2 (2.0–6.3 µmol/L), and in the index patient (2.4–4.7 µmol/L). All individuals had normal plasma free carnitine (data not shown).
Whenever possible, SCAD activity was measured both in fibroblasts and in lymphocytes. In the father, mother, and the index patient, SCAD activity was measured in both fibroblasts and lymphocytes. The fibroblast SCAD activity in the father and in the index patient was severely decreased. The lymphocyte SCAD activity showed strongly decreased levels: (0.03 and 0.07 nmol/min/mg protein, controls: 0.68 ± 0.13) in the index patient and his eldest sister, II.2. It was moderately decreased to 
0.3 nmol/min/mg protein in 1138C>T heterozygous individuals and to 0.5 nmol/min/mg protein (–1.3 standard deviation) in the 625A heterozygous individuals in this family.
SCAD DNA analysis of the family showed the presence of both the 1138C>T mutation and the 625G>A polymorphism in this family. The father was a compound heterozygote. The mother was heterozygous for 1138C>T, as were two of her children. One sibling (II.2) and the index patient were homozygous for 1138C>T. Homozygosity for 1138C>T in combination with 625A was not observed. Heterozygosity for 625A in 2 siblings showed normal EMA excretion and plasma C4-carnitine and slightly decreased SCAD activity in lymphocytes. Heterozygosity for 1138C>T in the mother and 2 siblings was associated with normal EMA excretion, normal plasma C4-carnitine, decreased SCAD activity in lymphocytes, but low-normal SCAD activity in fibroblasts of the mother. Double heterozygosity showed a borderline abnormal EMA excretion with elevated plasma C4-carnitine and decreased SCAD activity in lymphocytes and fibroblasts.
|
DISCUSSION |
|---|
|
TOPABSTRACTMETHODSDISCUSSIONREFERENCES |
|---|
SCAD (EC 1.3.99.2) is the first enzyme of the ß-oxidation of short-chain (C4) fatty acids. Only a limited number of cases have been reported in the literature thus far. In general, 2 clinical phenotypes may be distinguished, i.e., patients with episodic hypoglycemia and ketosis and patients with developmental delay and hypotonia.1 The index patient described here does not fit into one of these groups, his clinical presentation being characterized by transient infantile hepatic dysfunction following a premature delivery. The consequences of prolonged total parenteral nutrition cannot be ruled out.10 Ethylmalonic aciduria was discovered by chance during the selective screening of inborn errors of metabolism. A subsequent analysis of plasma acylcarnitines by tandem mass spectrometry showed a highly increased level of C4-carnitine. This strengthened the suspicion of SCAD deficiency. Confirmation of the enzyme defect was established by SCAD activity measurements in lymphocytes and fibroblasts, and SCAD gene sequence analysis showed homozygosity for the 1138C>T mutation. The 1138C>T mutation has been found to cause a fully inactive SCAD enzyme when overexpressed in Escherichia coli cells.1
This family study shows some aspects which expand the knowledge on SCAD deficiency and adds to its complexity. First, this is a clear report showing the possibility of neonatal SCAD deficiency screening as the index patient’s Guthrie screening card was examined for acylcarnitines half a year after delivery showing an elevated C4-carnitine. A limited number of patients with SCAD deficiency has been picked up by neonatal tandem mass spectrometric screening.11 However, there was no actual proof of a decreased enzyme activity or inactivating mutations in these patients.
Fetal fatty acid oxidation disorders causing life-threatening hepatic disease in mothers by fetal-maternal interaction, include long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency12 and CPT1 deficiency.13 Matern et al reported 1 case of SCAD deficiency with maternal acute fatty liver of pregnancy.6 Our report confirms the association between fetal SCAD deficiency and maternal disease and is the first report on maternal HELLP syndrome in SCAD deficiency. The fact that the pregnancy of the eldest surviving sibling had an unremarkable course indicates that maternal disease of pregnancy in SCAD deficiency is not an obligatory combination.
We studied the correlation between the genetic and various metabolic parameters in this family. Elevated urinary EMA excretion correlated well with increased plasma C4-carnitine, with strongly decreased SCAD enzyme activity in fibroblasts and lymphocytes, and a homozygous 1138C>T mutation. A virtually normal urine ethylmalonate was associated with compound heterozygosity, which, on the other hand, showed elevated C4-carnitine and a decreased SCAD enzyme activity. Plasma C4-carnitine was elevated in 1138C>T homozygotes and in compound heterozygotes showing the power of acylcarnitine analysis. In agreement with data from Corydon et al1 the assay of the residual SCAD activity in lymphocytes or cultured fibroblasts may give variable results. This is exemplified in our subjects I.1 and I.2 who were compound heterozygous and heterozygous respectively. Both had comparable lymphocyte activities, whereas the activity in the father’s (I.1) fibroblasts was more severely decreased. It may turn out that the true value of enzyme assays may be restricted to homozygotes for an inactivating mutation only. In this family SCAD activity was studied in lymphocytes and in fibroblasts. Markedly decreased SCAD activity (<20% of controls) accompanied homozygosity for the inactivating 1138C>T mutation, whereas heterozygotes for this mutation had enzyme activities around 40% of the mean of controls. These findings compare nicely with those observed in other simple autosomal recessively inherited disorders. Probably, the most reliable SCAD activity figures may be obtained by assaying SCAD in a muscle biopsy specimen. This approach was considered to be too invasive to apply in the present family.
As expected, heterozygotes for the common variant 625G>A had only a slightly decreased SCAD activity (70% of controls). Accordingly, there was a good correlation between SCAD enzyme activity determination and DNA analysis of the SCAD gene. But in the father we found different SCAD activity levels in fibroblasts and lymphocytes, which remains unexplained at present and warrants additional studies.
The SCAD gene may have so-called gene variations, 625G>A and 511C>T and/or pathogenic (inactivating) mutations, 268G>A, 575C>T, 973C>T, 310–312delGAG, 1058C>T, 1138C>T, or 1147C>T.1,5,14,15 In the literature one patient with an 1138C>T mutation has been described1; she presented during the first week of life with hypotonia and seizures and later showed a developmental delay. Her EMA level in urine was 45–68 mmol/mol creatinine, and a variably reduced (20%) SCAD activity was measured in fibroblast extracts. Mutation screening of the SCAD gene showed a 1138C>T mutation on one allele and a 625G>A on the second allele. Our study showed the father I.1 to have 1138C>T on 1 allele and 625G>A on the other allele. His urine EMA level was not elevated, his plasma C4-carnitine was elevated 4 times above the upper limit and his SCAD activity was decreased to 20% in fibroblasts and to 40% in lymphocytes, without any clinical sign or symptom ever. Thus far, all reported SCAD-deficient cases had clinical abnormalities, with one exception.16 The clinical symptoms were irrespective of the type of mutation, i.e., either inactivating mutations or polymorphisms. These polymorphisms were shown to result in thermolabile enzyme protein.7 The phenotypic expression of a given mutation is thought to depend on additional genetic17 or environmental determinants. Our results indicate that identification of mutations causing defective SCAD protein may not be predictive for the clinical expression of SCAD deficiency. However, the presence of mutations is the most important predisposing factor.1 This implies that mutation screening will continue to gain importance in family analysis. An index patient in any family will invariably be picked up by selective screening of metabolic disorders. The full power of neonatal screening of SCAD deficiency must still be proven. It is of utmost importance to describe and to define the natural history of SCAD deficiency to delineate the true need for treatment of this disorder.
Our studies contribute to the understanding that even severe inactivating SCAD mutations do not necessarily lead to clinical manifestations, given the existence of optimum external determinants. The nature of these external determinants remains to be established and needs to be resolved.
|
ACKNOWLEDGMENTS |
|---|
We gratefully acknowledge the expert assistance of J.P.N. Ruiter in performing the enzyme assays and S.M. Gersen-van Zadel in preparing the manuscript.
|
FOOTNOTES |
|---|
Received for publication Mar 22, 2002; Accepted Jan 30, 2003.
Address correspondence to Marinus Duran, PhD, Academic Medical Center, Department of Clinical Chemistry, Box 22660, 1100 DD Amsterdam, The Netherlands. E-mail: m.duran{at}amc.uva.nl
Deceased. 
Dr Corydon’s present address is Novozymes A/S, 2880 Bagsvaerd, Denmark.
|
REFERENCES |
|---|
|
TOPABSTRACTMETHODSDISCUSSIONREFERENCES |
|---|
- Corydon MJ, Vockley J, Rinaldo P, et al. Role of common gene variations in the molecular pathogenesis of short-chain acyl-CoA dehydrogenase deficiency. Pediatr Res.2001; 49 :18 –23[Web of Science][Medline]
- Amendt BA, Greene C, Sweetman L, et al. Short-chain acyl-coenzyme A dehydrogenase deficiency. Clinical and biochemical studies in two patients. J Clin Invest.1987; 79 :1303 –1309
- Bhala A, Willi SM, Rinaldo P, Bennett M, Schmidt-Sommerfeld E, Hale DE. Clinical and biochemical characterization of short-chain acyl-coenzyme A dehydrogenase deficiency. J Pediatr.1995; 126 :910 –915[CrossRef][Web of Science][Medline]
- Duran M, Walther FJ, Bruinvis L, Wadman SK. The urinary excretion of ethylmalonic acid: what level requires further attention? Biochem Med.1983; 29 :171 –175[CrossRef][Medline]
- Gregersen N, Winter VS, Corydon MJ, et al. Identification of four new mutations in the SCAD gene in two patients: one of the variant alleles 511C>T, is present at an unexpectedly high frequency in the general population, as was the case for the 625G>A, together conferring susceptibility to ethylmalonic aciduria.
Hum Mol Genet.1998; 7
:619
–627
[Abstract/Free Full Text] - Matern D, Hart P, Murtha A, et al. Acute fatty liver of pregnancy associated with short chain acyl-coenzyme A dehydrogenase deficiency. J Pediatr.2001; 138 :585 –588[CrossRef][Web of Science][Medline]
- Gregersen N, Andresen BS, Corydon MJ, et al. Mutation analysis in mitochondrial fatty acid oxidation defects: exemplified by acyl-CoA dehydrogenase deficiencies, with special focus on genotype-phenotype relationship. Hum Mutat.2001; 18 :169 –189[CrossRef][Web of Science][Medline]
- Vreken P, Bok LA, Gregersen N, et al. Biochemical and genetic studies in a family with short-chain acyl-CoA dehydrogenase (SCAD) deficiency. J Inherit Metab Dis.2000; 23(suppl 1) :124
- Vreken P, Lint van AEM, Bootsma AH, Overmars H, Wanders RJA, Gennip van AH. Rapid diagnosis of organic acidemias and fatty-acid oxidation defects by quantitative electrospray tandem-MS acylcarnitine analysis in plasma. Adv Exp Med Biol.1999; 466 :327 –337[Web of Science][Medline]
- Merritt RJ. Cholestasis associated with total parenteral nutrition. J Pediatr Gastroenterol Nutr.1986; 5 :9 –25[Web of Science][Medline]
- Naylor EW, Chace DH. Automated tandem mass spectrometry for mass newborn screening for disorders in fatty acid, organic acid, and amino acid metabolism. J Child Neurol.1999; 14(suppl 1) :S4 –S8
- Ibdah JA, Bennett MJ, Rinaldo P, et al. A fetal fatty-acid oxidation disorder as a cause of liver disease in pregnant women.
N Engl J Med.1999; 340
:1723
–1731
[Abstract/Free Full Text] - Innes AM, Seargeant LE, Balachandra K, et al. Hepatic carnitine palmitoyltransferase I deficiency presenting as maternal illness in pregnancy. Pediatr Res.2000; 47 :43 –45[Web of Science][Medline]
- Kristensen MJ, Bross P, Bhala A, Hale DE, Jensen TG, Gregersen N. New point mutations in short-chain acyl-CoA dehydrogenase. Am J Hum Genet.1993; 53 :915A
- Kristensen MJ, Kmoch S, Bross P, Andresen BS, Gregersen N. Amino acid polymorphism (Gly209Ser) in ACADS gene.
Hum Mol Genet.1994; 3
:1711
[Free Full Text] - Ribes A, Riudor E, Garavaglia B, et al. Mild or absent clinical signs in twin sisters with short-chain acyl-CoA dehydrogenase deficiency. Eur J Pediatr.1998; 157 :317 –320[CrossRef][Medline]
- Dipple KM, Phelan JK, McCabe ERB. Consequences of complexity within biological networks: robustness and health, or vulnerability and disease. Mol Genet Metab.2001; 74 :45 –50[CrossRef][Web of Science][Medline]
PEDIATRICS (ISSN 1098-4275). ©2003 by the American Academy of Pediatrics
CiteULike 
Connotea 
Del.icio.us 
Digg 
Facebook 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  B. T. van Maldegem, M. Duran, R. J. A. Wanders, K. E. Niezen-Koning, M. Hogeveen, L. Ijlst, H. R. Waterham, and F. A. Wijburg Clinical, biochemical, and genetic heterogeneity in short-chain acyl-coenzyme A dehydrogenase deficiency. JAMA, August 23, 2006; 296(8): 943 - 952. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
